| Objectives: Periodontal ligament stem cells(PDLSCs)have broad application prospects in bone tissue engineering because of their strong self-renewal ability and multilineage differentiation potential,and this project takes PDLSCs as the research object to explore the effect of in vitro stretch stress on the expression of Girdin in periodontal stem cells.In order to further elucidate a series of adaptive modification processes in periodontal tissues under the action of orthodontic correction force,as well as the related mechanisms and principles involved,it also provides a theoretical basis for exploring the biomechanical mechanism of intracellular transduction of mechanical force signals.Methods:1.Periodontal membrane stem cells were cultured in vitro by modified tissue block attachment method,and passaged to PDLSCs,and PDLSCs were identified by multidirectional differentiation induction experiment,flow cytometry technology and immunofluorescence technology;2.The Forcel four-point curved cell mechanical loader was used to apply dynamic tension stress to the cells,Ed U test,CCK-8 test and other methods were used to detect the change of their proliferation ability after stretching force was applied to periodontal membrane stem cells,scratch test was used to detect the change of migration efficiency after stretching force was added to periodontal membrane stem cells,and Western blot method was used to detect the expression level of Girdin and P-girdin proteins in periodontal membrane stem cells by dynamic tensile tension.The effect on the expression level of osteogenesis-related genes in periodontal stem cells was observed;3.The synthetic si RNA sequence was used to interfere with the Girdin protein in periodontal membrane stem detected by Western Blot method in periodontal membrane stem cells before and after interference The protein expression was detected by the effect of silencing Girdin on the osteogenic differentiation ability of periodontal membrane stem cells;4.The synthetic si RNA sequence was used to interfere with the expression of Girdin protein in periodontal membrane stem cells,and then the stretching force was applied to detect the expression of Girdin and PGirdin before and after interference and afterburner,as well as the expression level of osteogenic genes and proteins(RUNX2,ALP,COL1)of PDLSCs.Results:1.The PDLSCs cultured in this experiment are typical fibroblast-like morphology,and have the multi-directional differentiation ability of osteogenesis and lipogenesis,and the corresponding detection by flow cytometry shows that both cells actively express CD29,CD44,and negative expression CD34 and CD45,which indicates that the periodontal membrane stem cells cultured in this experiment have the characteristics of mesenchymal stem cells;2.Immunofluorescence laser confocal microscopy showed that Girdin protein was mainly distributed in the cytoplasm of dental PDLSCs,and P-Girdin protein was mainly distributed in the nucleus of PDLSCs;3.Ed U experiments showed that the expression of knocking down Girdin significantly inhibited the proliferation of PDLSCs,and the application of tension promoted the proliferation of PDLSCs,and the proliferative ability of periodontal membrane stem cells after knocking down Girdin was significantly enhanced compared with that of the group with simple application of stretch,and the results of CCK-8test showed that knocking down Girdin protein could significantly inhibit the proliferation of periodontal membrane stem cells.Scratch experiments showed that the expression of silencing Girdin protein also had an inhibitory effect on the migration ability of periodontal membrane stem cells,and osteogenic differentiation induction experiments showed that silencing Girdin protein showed an inhibitory trend on the formation of mineralized nodules of periodontal membrane stem cells;4.The results of Western blot experiment showed that after the application of traction,the protein expression levels of Girdin,P-Girdin and osteogenic differentiation-related proteins(RUNX2,ALP,COL1)showed a significant upward trend,knocking down Girdin did not apply stretch stress,and the protein expression levels of Girdin and P-Girdin osteogenic differentiation-related proteins(RUNX2,ALP,COL1)showed a significant downward trend.The protein expression levels of Girdin,P-Girdin,and osteogenic differentiation-related proteins(RUNX2,ALP)after knocking down Girdin were higher than those in the control group and the knockdown Girdin no afterburner group.Conclusions:1.Under the action of suitable mechanical stress,Girdin participated in the mechanical stress signaling process in PDLSCs,and its expression showed an increasing trend under the action of suitable orthodontic stress;2.Girdin protein is an important factor involved in regulating the proliferation,migration and osteogenic differentiation of PDLSCs. |
